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52 Cards in this Set
- Front
- Back
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What is a nanosensor? |
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What advantages do nanosensors offer over traditional assays? |
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Why is ratiometric analysis important? |
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What are the delivery systems into intracellular environments? |
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What is importunate about the size of a biosensor/nanosensor? |
When penetrating individual live cells, even the introduction of a sub-micron sensor tip can cause biological damage and resultant biochemical consequences. So the sensor needs to be small enough to not change the biological function of the system. |
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What are the complications of using fluorescent dyes when observing the structure of a cell? |
- The fluorescent dyes can be toxic to the cell. - The fluorophore can cause damage to the cell and derange its normal behaviour - So, we are not sure if the abnormal behaviour we are seeing is part of a pathological process or due to the toxicity of the introduced fluorophore. |
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What are the 2 types of nanosensor? |
Active Passive |
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Give 7 advantages of magnetic nanosensing protein detection? |
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What are 3 previous technologies for general diagnosis? |
- Biopsy - Blood detection - Clinical imaging |
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Define biomarker? |
an indicator of a biological state or condition. It can be a protein, a fragment of a protein, DNA/RNA, or an organic chemical made by abnormal cells. |
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What are the traditional diagnostic methods for cancer? |
- endoscopy, - computed tomography, - X-rays, positron emission tomography, - mammography and - magnetic resonance imaging |
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What are some problems with traditional diagnostic cancer methods? |
- Not accessible to large populations - Not practical for repeated screenings at early stages of disease |
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Give a brief overview of ELISA as a high throughput screening method for detecting biomarkers. |
It involves specific antibodies for a particular biomarker and signals its detection by a chromogenic reporter and substrate. |
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What are 3 disadvantages of ELISA? |
- time-consuming, - technically burdensome, - costly |
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What are PEBBLEs? |
probes encapsulated by biologically localised embedding that allow for minimally intrusive sensing of ions in cellular environments due to their small size and protect the sensing elements (i.e. fluorescent dyes) by encapsulating them within an inert matrix. |
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What size are PEBBLEs? |
20 to 600 nm in diameter |
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What is an advantage of PEBBLEs? |
- The protective coating eliminates interference such as protein binding or membrane/organelle sequestration which alter dye response. - The nanosensor matrix enables dyes that would usually be toxic to the cell to be used for intracellular sensing since the matrix provides protection to the cellular contents. |
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What analytes have been measured with PEBBLEs? |
- calcium - potassium - NO - oxygen - chloride - sodium - glucose |
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What was the first technology designed to measure intracellular space? |
Fibre-based optical nanosensors (optical fibre optodes) |
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What is the size of an optical fibre optode? |
The tip was very small (100 nm) but they quickly become larger up the taper |
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How did an optical fibre optodes work? |
The fibre optic tip was a pH responsive fluorophore coated with PVC. It measured the change in pH causing a change in fluorescence which was sent along the optical fibre, amplified and read. |
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What were the problems with the optical fibre optode nanosensors? |
The size of the taper meant multiple insertions into the cell can damage it, so it was not responsive in its natural state. |
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What nanosensor was developed from the optical fibre optodes? |
pulled fibre technology |
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What nanosensor is derived from pulled fibre technology? |
PEBBLEs - fluorescent nanosensor
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What was the key developments of PEBBLE and where do they originate from? |
Derived from pulled fibre technology which is derived from optical fibre optodes - PEBBLE enable a discrete observation platform - the sensor can be integrated into the cell in large numbers |
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What are the key characteristics of PEBBLEs? |
- 20 nm radius (1 parts per billion ppb of cell volume) making them easy to introduce into cells - the cell viability remains high 97% - the response time is <1 ms - a wide number of analytes can be measured |
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What materials can PEBBLEs matrices be made from? |
- polyacrylamide - cross-linked decyl methacrylate, - silica-based sol-gel - spacious, good for entrapping enzymes as it will not distort the structure of the enzyme Each matrix can be combined with specific "free dyes", ionophores, or enzymes to produce sensors selective for the biological component of interest |
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What is a ratiometric device? |
measures the ratio between two fluorescence intensity values that are taken at two excitation wavelength. It can correct for unequal dye loading |
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What is Sol-gel PEBBLE used to measure and how does it do this? |
Intracellular oxygen It is a ratiometric device - The Ruthenium dye fluorescence is quenched very efficiently in the presence of oxygen, so its fluoressence intensity goes down with greater local oxygen concentration - The Oregon green dye fluorescence is not sensitive to oxygen, so its intensity stays the same and is used as a reference. |
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What changes the intensity of the fluorescence of the 2 dyes in a ratiometric PEBBLE device? |
- varying amount of PEBBLE present in solution - changes in intensity of the excitation light but the ratio between the active dye and the reference dye remain the same and this is what is being measured |
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How has interference been compared between free fluorescent dyes and PEBBLEs? |
When measuring Ruthenium:Oregon green fluorescence, the ratio of Ru:OG goes up in the free dye solution with increasing concentration of BSA, whereas the ratio stays the same on the Sol-gel PEBBLEs this is the desired result since the amount of oxygen in the solution is not changing. - the ratio has been maintained because the dye is encapsulated in the porous shell, protecting it from the effect of protein. The analyte can still get in and out through the pores of the nanosensor and interact with the fluorophore, but because the protein is large, with a high MW the BSA cannot. |
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What is the most important consideration when choosing a delivery methods for PEBBLE sensor insertion? |
Methods of sensor insertion can be specific to the cell type used for experimentation and many factors play a role in the choice, most importantly whether the cells in culture are adherent. |
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Who developed pulled fibre technology? |
Tan et al. |
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What are the disadvantages of optical fibre optodes? |
- Invasive to the cellular system (taper size) - Technique requires highly skilled operators to position the probe in the cell - Even though the optode has submicron diameters the penetrations are still >1% of the cell volume, which could induce severe perturbations, limiting cellular viability |
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What are the disadvantages of fluorescent probes? |
- easier to use than optical fibre optodes but still perturb the cell. - can be cytotoxic - they have variations in signal intensity due to the non-specific protein binding, organelle sequestration, efflux of dye by membrane proteins - fluorescent dyes have limited experimental time once the fluorophore is loaded in the cell |
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What are the advantages of the sensor platform in PEBBLEs? |
- it can provide a convenient cage that facilitates the entrapment of a number of sensing components, and access to just small analytes, offering the ability to fabricate ratiometric sensors from complementary probes, the senors can be complex and they will not get interference as other substances with big MW eg proteines etc cant enter the cage. |
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Why is the delivery method of PEBBLE sensors so important? |
- important to retain cellular viability - suitable amount of sensor must be loaded into the cell to ensure an adequate fluorescent signal - critical in a long term experimentation is the cells are still dividing as this will effectively reduce the amount of sensors within the cell at each division. |
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What are some methods of PEBBLE delivery into cells? |
- Gene gun offers high loading of PEBBLE sensors for adherent cells and is the best method
- CPP functionalization has proved effective for cell cultures generally with varying levels of sensor loading, depending on the cell type - Lipid transfection gives good results once conc of transfection reagent and sensor loading are established - Picoinjection allows location specific PEBBLE delivery but the loading is not as high so it is more suited to larger cells |
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What is the consideration regarding positioning with the Gene gun method? |
- there is no control over the positioning of thesensors although once the correct pressure and particle concentrationsare found, this method is very effective for the delivery ofmany sensors in a single blast with a fairly even spread ofsensors throughout the cell |
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What is the cell viability when using the Gene gun method? |
98% |
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Why is the Gene gun only suitable for adherent cell cultures? |
The media must be removed prior to sensor bombardment |
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Give some examples of cells that the Gene gun has been used in for inserting PEBBLE? |
A range of nanosensors have been inserted into many different adherent cell types (with high sensor loading, and good cell viability): - astrocytes - hepatocytes - CHO cells - several epithelial cell lines |
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What is a way that free radicals could be measured using nanosensors? |
Phagocytosis When a macrophage comes into contact with a foreign compound, it will engulf it. Groups of nanosensors can be internalised into macrophage vacuoles via routine cell processes. The nanosensors are then encapsulated in phagosomes, they are then bombarded with free radicals, dyes that are sensitive to ROS measure the amount. |
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What are polyacrylamide sensors synthesised in? |
Water-in-oil microemulsion system |
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What are the properties of a water-in-oil microemulsion system? |
- transparent - isotropic - thermodynamically stable liquid media - continuous oil domain - aqueous domain thermodynamically compartmentalised by a surfactant |
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What is the water-in-oil microemulsion sometimes referred to as? |
revers micellar solution/droplet |
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What main 2 events take place in the aqueous core of the micellar droplet? |
- The polymerisation reaction - Formation of nanoparticles |
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What is the size of the synthesised polyacrylamide PEBBLEs and why? |
nanometer range The PEBBLE components are hydrophilic so stay in the aqueous core where both the reaction and formation occurs. |
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How can the size of the polyacrylamide nanospheres be varied? |
By changing the amount of - surfactant - solvent - monomer - temperature of the reaction |
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What is the reproducibility of polyacrylamide matrix PEBBLEs? |
Very good, once a procedure is optimised the protocol will be reliable for producing the required sensors Inter and intra batch reproducibility is consistent but each batch should be calibrated to ensure this |
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What applications have been used by polyacrylamide sensors so far? |
- pH - calcium - oxygen - glucose - zinc - magnesium |
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What determines the size of the PEBBLE and how? |
The initial size of the aqueous core of the micellar droplet by encapsulating the reactions and preventing the sphere from becoming too large during polymerisation |
